Limb functional recovery is impaired in fibroblast growth factor-2 (FGF2) deficient mice despite chronic ischaemia-induced vascular growth.

FGF2 is a potent stimulator of vascular growth; however, even with a deficiency of FGF2 (Fgf2-/-), developmental vessel growth or ischaemia-induced revascularization still transpires. It remains to be elucidated as to what function, if any, FGF2 has during ischaemic injury. Wildtype (WT) or Fgf2-/- mice were subjected to hindlimb ischaemia for up to 42 days. Limb function, vascular growth, inflammatory- and angiogenesis-related proteins, and inflammatory cell infiltration were assessed in sham and ischaemic limbs at various timepoints. Recovery of ischaemic limb function was delayed in Fgf2-/- mice. Yet, vascular growth response to ischaemia was similar between WT and Fgf2-/- hindlimbs. Several angiogenesis- and inflammatory-related proteins (MCP-1, CXCL16, MMPs and PAI-1) were increased in Fgf2-/- ischaemic muscle. Neutrophil or monocyte recruitment/infiltration was elevated in Fgf2-/- ischaemic muscle. In summary, our study indicates that loss of FGF2 induces a pro-inflammatory microenvironment in skeletal muscle which exacerbates ischaemic injury and delays functional limb use.

mTOR Kinase Inhibition Effectively Decreases Progression of a Subset of Neuroendocrine Tumors that Progress on Rapalog Therapy and Delays Cardiac Impairment.

Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432-41. ©2017 AACR.

The role of proximal versus distal stomach resection in the weight loss seen after vertical sleeve gastrectomy.

The mechanisms involved in the weight loss seen after vertical sleeve gastrectomy (VSG) are not clear. The rat stomach has two morphologically and functionally distinct proximal and distal parts. The rat model for VSG involves complete removal of the proximal part and 80% removal of the distal part along the greater curvature. The purpose of this study was to understand the potential independent contributions of removal of these distinct gastric sections to VSG outcomes. We prepared four surgical groups of male Long-Evans rats: VSG, sham surgery (control), selective proximal section removal (PR), and selective distal section removal (DR). Gastric emptying rate (GER) was highest after VSG compared with all other groups. However, PR, in turn, had significantly greater GER compared with both DR and sham groups. The surgery-induced weight loss followed the same pattern with VSG causing the greatest weight loss and PR having greater weight loss compared with DR and sham groups. The results were robust for rats fed regular chow or a high-fat diet. Body mass analysis revealed that the weight loss was due to the loss of fat mass, and there was no change in lean mass after the surgeries. In conclusion, removal of the proximal stomach contributes to most, but not all, of the physiological impact of VSG.

Optical and nuclear imaging of glioblastoma with phosphatidylserine-targeted nanovesicles.

Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Modeling pulmonary alveolar microlithiasis by epithelial deletion of the Npt2b sodium phosphate cotransporter reveals putative biomarkers and strategies for treatment.

Pulmonary alveolar microlithiasis (PAM) is a rare, autosomal recessive lung disorder associated with progressive accumulation of calcium phosphate microliths. Inactivating mutations in SLC34A2, which encodes the NPT2b sodium-dependent phosphate cotransporter, has been proposed as a cause of PAM. We show that epithelial deletion of Npt2b in mice results in a progressive pulmonary process characterized by diffuse alveolar microlith accumulation, radiographic opacification, restrictive physiology, inflammation, fibrosis, and an unexpected alveolar phospholipidosis. Cytokine and surfactant protein elevations in the alveolar lavage and serum of PAM mice and confirmed in serum from PAM patients identify serum MCP-1 (monocyte chemotactic protein 1) and SP-D (surfactant protein D) as potential biomarkers. Microliths introduced by adoptive transfer into the lungs of wild-type mice produce marked macrophage-rich inflammation and elevation of serum MCP-1 that peaks at 1 week and resolves at 1 month, concomitant with clearance of stones. Microliths isolated by bronchoalveolar lavage readily dissolve in EDTA, and therapeutic whole-lung EDTA lavage reduces the burden of stones in the lungs. A low-phosphate diet prevents microlith formation in young animals and reduces lung injury on the basis of reduction in serum SP-D. The burden of pulmonary calcium deposits in established PAM is also diminished within 4 weeks by a low-phosphate diet challenge. These data support a causative role for Npt2b in the pathogenesis of PAM and the use of the PAM mouse model as a preclinical platform for the development of biomarkers and therapeutic strategies.

Combining micro-computed tomography with histology to analyze biomedical implants for peripheral nerve repair.

BACKGROUND: Biomedical implants used in tissue engineering repairs, such as scaffolds to repair peripheral nerves, can be too large to examine completely with histological analyses. Micro-computed tomography (micro-CT) with contrast agents allows ex vivo visualization of entire biomaterial implants and their interactions with tissues, but contrast agents can interfere with histological analyses of the tissues or cause shrinkage or loss of antigenicity. NEW METHOD: Soft tissue, ex vivo micro-CT imaging using Lugol's iodine was compatible with histology after using a rapid (48 h) method of removing iodine. RESULTS: Adult normal and repaired rat sciatic nerves were infiltrated ex vivo with iodine, imaged with micro-CT and then the iodine was removed by incubating tissues in sodium thiosulfate. Subsequent paraffin sections of normal nerve tissues showed no differences in staining with hematoxylin and eosin or immunostaining with multiple antibodies. Iodine treatment and removal did not alter axonal diameter, nuclear size or relative area covered by immunostained axons (p>0.05). Combining imaging modalities allowed comparisons of macroscopic and microscopic features of nerve tissues regenerating through simple nerve conduits or nerve conduits containing a titanium wire for guidance. COMPARISON WITH EXISTING METHODS: Quantification showed that treatment with iodine and sodium thiosulfate did not result in tissue shrinkage or loss of antigenicity. CONCLUSIONS: Because this combination of treatments is rapid and does not alter tissue morphology, this expands the ex vivo methods available to examine the success of biomaterial implants used for tissue engineering repairs.

Preclinical pharmacological evaluation of letrozole as a novel treatment for gliomas.

We present data that letrozole, an extensively used aromatase inhibitor in the treatment of estrogen receptor-positive breast tumors in postmenopausal women, may be potentially used in the treatment of glioblastomas. First, we measured the in vitro cytotoxicity of letrozole and aromatase (CYP19A1) expression and activity in human LN229, T98G, U373MG, U251MG, and U87MG, and rat C6 glioma cell lines. Estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cells served as controls. Cytotoxicity was determined employing the MTT assay, and aromatase activity using an immunoassay that measures the conversion of testosterone to estrogen. Second, in vivo activity of letrozole was assessed in Sprague-Dawley rats orthotopically implanted with C6 gliomas. The changes in tumor volume with letrozole treatment (4 mg/kg/day) were assessed employing µPET/CT imaging, employing [(18)F]-fluorodeoxyglucose (F18-FDG) as the radiotracer. Brain tissues were collected for histologic evaluations. All glioma cell lines included here expressed CYP19A1 and letrozole exerted considerable cytotoxicity and decrease in aromatase activity against these cells (IC50, 0.1-3.5 µmol/L). Imaging analysis employing F18-FDG µPET/CT demonstrated a marked reduction of active tumor volume (>75%) after 8 days of letrozole treatment. Immunohistochemical analysis revealed marked reduction in aromatase expression in tumoral regions of the brain after letrozole treatment. Thus, employing multifaceted tools, we demonstrate that aromatase may be a novel target for the treatment of gliomas and that letrozole, an FDA-approved drug with an outstanding record of safety may be repurposed for the treatment of such primary brain tumors, which currently have few therapeutic options.

In vivo optical imaging of brain tumors and arthritis using fluorescent SapC-DOPS nanovesicles.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

Ocular delivery of pRNA nanoparticles: distribution and clearance after subconjunctival injection.

PURPOSE: RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA of bacteriophage phi29 DNA packaging motor were previously found to be thermodynamically stable. As the nanoparticles could have potential in ocular drug delivery, the objectives in the present study were to investigate the distribution of pRNA nanoparticles after subconjunctival injection and examine the feasibility to deliver the nanoparticles to the cells of cornea and retina. METHODS: Alexa647-labeled pRNA nanoparticles (pRNA-3WJ and pRNA-X) and double-stranded RNA (dsRNA) were administered via subconjunctival injection in mice. Alexa647 dye was a control. Topical administration was performed for comparison. Ocular clearance of pRNA nanoparticles and dsRNA after the injection was assessed using whole-body fluorescence imaging of the eyes. The numbers of cells in the ocular tissues with nanoparticle cell internalization were determined in fluorescence microscopy of dissected eye tissues. RESULTS: After subconjunctival injection, pRNA nanoparticles and dsRNA were observed to distribute into the eyes and cleared through the lymph. pRNA-3WJ, pRNA-X, and dsRNA were found in the cells of the conjunctiva, cornea, and sclera, but only pRNA-X was in the cells of the retina. Topical administration was not effective in delivering the nanoparticles to the eye. CONCLUSIONS: The pRNA nanoparticles were delivered to the cells in the eye via subconjunctival injection, and cell internalization was achieved in the cornea with pRNA-3WJ and pRNA-X and in the retina with pRNA-X. Only the X-shape pRNA-X could enter the retina.

Molecular strategy to reduce in vivo collagen barrier promotes entry of NCX1 positive inducible pluripotent stem cells (iPSC(NCX¹âº)) into ischemic (or injured) myocardium.

OBJECTIVE: The purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium. BACKGROUND: We previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium. METHODS: In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSC(NCX1+)), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly. RESULTS: The number of green fluorescent protein positive (GFP(+)) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling. CONCLUSION: Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSC(NCX1+) penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.